Xenopus sperm chromatin preparation

Sperm chromatin is the substrate for nuclear assembly and can be readily prepared after sperm collection from the testes of well-fed freshly killed adult male Xenopus. Two animals contain enough sperm for more than 50 experiments. Sperm should be demembranated with lysolecithin exactly as described by Lohka and Masui (1984). After demembranation, sperm heads (chromatin) should be aliquoted at about 2.4 x IdV/itl in a buffer containing 30% (v/v) glycerol, 200 mM sucrose, 2.4 mM MgCl , and 20 mMNa-maleate, pH 6.8. Once aliquoted into small polypropylene tubes, tubes should be immersed in liquid nitrogen and stored at -70°C. Xenopus sperm chromatin prepared in this way can be stored for at least 5 years without apparent deterioration. (For additional details, see the chapter by Lohka, this volume.)... 

Prepare Xenopus egg extracts (Newmeyer and Wilson, 1991) and incubate extract with demembranated Xenopus sperm chromatin to assemble nuclei. ER and Golgi will also assemble in the same extract. 

Although several different methods exist for the preparation of Xenopus sperm nuclei, all of these protocols share the essential step of membrane permeabilization. The sperm cells from other species may, however, require additional treatments before they can be used in remodeling studies. For example, mammalian sperm chromatin is stabilized by disulfide bonds between protamine molecules (for review, see Perreault, 1992), and it is clear that reduction of these bonds... 

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